The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones.

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4) 2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)(2) tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication.